The long-term objective of this project is to identify new, more effective, and less toxic therapies for Head and Neck Squamous Cell Carcinoma (HNSCC) and related cancers, by understanding the function and regulation of the p53 family members p63 and p73. The p53 family of proteins plays a key role in the pathogenesis of HNSCC. The p53 gene itself is a target of mutational inactivation in more than 50% of these tumors, and p53-mutant tumors are highly lethal regardless of treatment modality. In contrast, the related family member p63 is not mutated but is dramatically overexpressed and/or subject to genomic amplification in the majority of cases. A contribution of p63 to HNSCC is further supported by the essential role we and others have demonstrated for p63 in promoting proliferation, adhesion signaling, and regenerative potential during normal epithelial development. In HNSCC cells p63 also functions as a repressor of apoptosis mediated by the related family member p73. Pro-apoptotic p73 itself is overexpressed particularly in p53-mutant HNSCC, and p63 suppresses apoptosis in these tumors through both physical association with p73 and direct binding to regulatory elements within p73-regulated pro- apoptotic genes. The physiologic significance of these observations is supported by the demonstration that p63/p73 are direct mediators of chemosensitivity in HNSCC which is abrogated by Bcl-2 up-regulation, a mediator of therapeutic resistance. Taken together, these findings provide a strong rationale for further studies to understand the biochemical regulation and functional contribution of p63/p73 in HNSCC. This proposal describes a systematic approach to uncovering the regulation of p63/p73 and their role in tumor maintenance in vivo. We hypothesize that a subset of p63/p73 regulators functions as endogenous apoptosis suppressors in HNSCC. We have conducted a genome wide-screen to identify such regulators, complemented by biochemical studies to identify direct p63-associated transcriptional regulators. In Aim 1 we will perform proof-of-principle studies for targeting chromatin in HNSCC based on our identification of essential p63 transcriptional co-factors. In Aim 2 we will validate additional p63-associated co-factors which we hypothesize to mediate distinct transcriptional programs relevant to tumor progression. In Aim 3 we will test the contribution of these transcriptional programs directly using a genetically engineered mouse HNSCC model. In Aim 4 we will focus on mining our original screen for new p63-regulators, performing direct validation of the most attractive candidates, correlating their expression with clinical outcome in primary tumor specimens, and performing biochemical studies to determine their mechanism. In addition to improving our knowledge of the basic biology of HNSCC, these studies will advance the goal of uncovering novel and viable therapeutic targets to improve treatment outcomes in this disease.